Nitrous acid treatment for separating impurities from antipernicious anaemia active material



Patented Mar. 3, 1953 NITROUS ACID TREATMENT FOR SEPARAT- ING IMPURITIES FROM ANTIPERNICIOUS ANAEMIA ACTIVE MATERIAL Ernest Lester Smith, North Harrow, and Stanley Ball, London, England, assignors to Glaxo Laboratories Limited, Greenford, 'Middlesex, England, a British company NoDrawing. Application March 27, 1950, Serial No. 152,272. In Great Britain July 13, 1949 14 Claims. 1

This invention is concerned with improvements in and relating to the manufacture of preparations containing material having anti pernicious anaemia activity.

The anti-pernicious anaemia factor is believed tocontain two or possibly more separate and distinct active compounds, one of which has recently become known as Vitamin B12. The term active material as used herein means any one compound or a mixture of two or more separate and distinct compounds having anti pernicious anaemia activity as determined by clinical trials, irrespective of the number of such compounds and irrespective of the source or sources of the material.

Active material may be obtained from liver, and various methods of obtaining extracts rich in active material from liver have been proposed. More recently it has become known that active material is produced in a suitable culture medium by certain micro-organisms, an example of which is Streptomyces grz'seus. When anti-pernicious anaemia factors are produced microbiologically, they are not always present in the culture medium in a form which shows anti-pernicious anaemia activity, and which is suitable for further purification, and in such cases it may be necessary to apply one or more of the processes described in application Serial No. 93,205 filed May 13, 1949, now abandoned in order to obtain active material. The last mentioned specification also describes how micro-organism which produce antipernicious anaemia factors may be recognized.

The present invention is concerned with the removal of impurities from extracts containing active material as above defined. For the better understanding of the present specification, it should be noted that whether or not a given material has anti-pernicious anaemia activity can only be definitely determined by clinical trials. Gn the other hand an examination of its microbiological activity by the so-called cup-plate assay, using Lactoba-cillus lactis Domer r Lactobacillus Zeichmam'i, and using pure Vitamin B12 as a standard (see e. g. Cuthbertson, Biochem J 1949, Proc. V.) will give an indication of antipernicious anaemia activity which will generally be reliable, lhe term Vitamin B12 activity as used hereinafter means activity of a given material measured microbiologically as described, as distinct from activity determined by clinical tests.

Active material derived from liver or from micro-organisms is usually accompanied by impurities, some of which are amino-acids and peptides which, being amphoteric, are difiicult to separate. It is the object of the present invention to provide a process whereby crude extracts of active material may be freed from such impurities, without loss of anti-pernicious anaemia activity.

We have found that, surprisingly, active material does not lose its anti-pernicious anaemia activity when treated with nitrous acid. We have found further that if an extract containing active materials and amino-acid and peptide impurities is treated with nitrous acid, the said impurities are converted into hydroxyacids, which may be much more readily separated from the active ma terial, either by removing the acids from the mixtures, or by the use of a solvent which will extract the active material itself.

According to the present invention, we provide a process for removing impurities from an extract containing active material as herein defined in which the said extract is treated with nitrous acid. The resulting solution may thereafter be treated in such a way as to bring about a separation between the active material and the acid impurities present in the solution.

According to a further feature of the invention, after the treatment with nitrous acid, the acidic impurities are separated from the solution.

According to another feature of the invention the solution after treatment with nitrous acid is rendered neutral or alkaline and the active material contained in the solution extracted there..- from by means of a suitable solvent as herein defined.

The aqueous extracts used as starting material in the process according to the invention can be of any desired degree of purity or concentration provided that they show anti-pernicious anaemia activity. Thus for example, We may use a crude aqueous extract of liver or a crude extract prepared from micro-organisms, for example by heat treatment or proteolysis of the culture liquors as described in the said application .Serial No. 93,205 or alternatively one or more purification treatments, such for example, as absorption on charcoal, solvent extraction and the like may be applied before use of the process according to the invention.

The treatment with nitrous acid can be carried out by any convenient method; thus for example we may acidify the aqueous extract of the active material and add a nitrite such for example as sodium nitrite. Alternatively, for example, the extract may be treated with nitrous fumes, or the nitrous acid generated from an alkylnitrite such as amylnitrite. I

Since active material may be destroyed by strongly acid conditions it will be obvious that treatment with nitrous acid should be carried out under such conditions that the active material is not destroyed. Generally we have found that treatment at a pH within the range of from 1 to 5 gives satisfactory results.

In practice we prefer to acidify the aqueous extract to about pH 4 with an organic acid, for example acetic acid or a mineral acid, for example hydrochloric acid and treat the resulting solution with sodium or potassium nitrite, further portions of acid being added if necessary to main.- tain the pH at about 4; it will be understood however that any other method of treatment with nitrous acid may be used provided that strongly acid conditions such as would destroy active material are avoided.

The nitrous acid is preferably used in excess and in order that this may be done the amino groups in the starting material should be assayed, for example by the well-known method of Van Slyke. When the quantity of nitrous acid required for the reaction is known, the quantities of the reagents are so adjusted as to give an excess of the nitrous acid for example by the use of an excess of the sodium nitrite where this is used. When the reaction has been completed the excess of nitrous acid may, if desired, be destroyed, for example by the addition of urea.

The treatment with nitrous acid may be carried out at room temperature, but in general, we prefer gently to warm the reaction mixture, it should however be understood that too high a temperature of reaction may cause destruction of the active material.

As stated above the treated inpurities may be separated from the active material in two ways; thus the impurities may be extracted from the solution resulting from the nitrous acid treatment or alternatively the active material itself may be extracted from this solution. Obviously both these techniques may be used in combination.

Where it is desired to extract the acidic impurities from the solution, methods of extracting hydroxy acids from aqueous solutions containing them may be used. Thus for example the solution resulting from the nitrous acid treatment may be extracted under acid conditions with an organic solvent capable of dissolving hydroxy acids. Many organic solvents may be used for this purpose but at present we have found the following solvents to be particularly suitable: ether, chloroform, lower aliphatic esters, butyl and amyl alcohols. These solvents are all at least partiallyimmiscible with water. Certain solvents in particular butyl and amyl alcohols, may also extract part of the active material and it is accordingly generally desirable to extract the solvent extract itself with a quantity of water in order to recover any active material which may have been extracted by the solvent.

The solvent extraction-of the acids should not be carried out under strongly acid conditions which would destroy the active material; at present we prefer to carry out this extraction at a pH within the range of from 1-5.

Other methods of extraction of acids which may be used include the use of anion-exchange resins, such as Deacidite or the Amberlite resins IRA. 400 and IR-4B, precipitation with metallic salts and the like.

Where it is desired to extract the active material from the solution resulting from the nitrous acid treatment, the latter may be rendered neutral or alkaline and the resulting solution extracted with a suitable solvent as herein defined.

The term a suitable solvent as used herein means an alcohol soluble in water at room temperature to the extent of from 3 to 25% by volume or a mixture of such alcohols, mixtures of phenol and/ or one or more of the cresols with any of these alcohols, mixtures of phenol and/or one or more of the cresols with not more than three times their weight of any ketone or ester soluble in water at ordinary temperatures, to the extent of from 3 to 25% by volume, phenol and/or one or more of the cresols mixed with not more than three times their weight of di-ethyl ether, di-isopropyl ether, benzene, toluene and/ or xylene, phenol and/or one or more of the cresols mixed with sufficient water to liquefy them, propyl or isopropyl alcohol containing from 5-25% by volume of water.

Where aqueous propyl and isopropyl alcohols are used as the suitable solvent the aqueous solution should contain a sumcient quantity of a salting-out agent to prevent complete miscibility.

At present we prefer to use phenol or a mixture of phenol and butanol as the suitable solvent. The reaction mixture may be rendered neutral or alkaline by the use of any convenient reagent, but at present we prefer to use sodium or potassium hydroxide or carbonate; it should however be noted that strongly alkaline conditions are likely to cause destruction of the active material and we accordingly prefer that the pH of the mixture should not exceed 9 during this extraction.

The process according to the invention may, if desired be followed by any convenient steps of purification but it should be mentioned that'the process forms a very useful partial purification step as referred to in the said application Serial No. 93,205.

In order that the invention may be well understood the following examples are given only as illustrations.

Example 1 50 ml. of a partially purified aqueous solution of active material (prepared from the broth obtained by the growth of Streptomyces grz'seus, by the heat treatment described in application Serial No. 93,205 followed by partial purification by adsorption onto charcoal, elution with phenol containing 10% of water and removal of the phenol by treatment with ether and water) was acidified to a pH of 4 with glacial acetic acid and 5 ml. of saturated sodium nitrite solution added. The mixture was warmed to about for 5 minutes and then allowed to cool for 20 minutes.

The cold solution was extracted 3 times with equal volumes of chloroform and then twice with equal volumes of butanol. The butanol solutions were back-washed with a little water, the water washings being added to the main aqueous solution. This Water extract was then adjusted to pH 7.5 by means of solid sodium bicarbonate and extracted 3 times with butanol-phenol mixture (3:1 v./v.).

The active material contained in the butanolphenol extract was transferred into aqueous solution by adding 5 volumes of diethyl ether and Washing the phenol-ether layer with water. All the phenol was removed from the aqueous solution by washing further with ether and the final solution containing the active material was light brown in colour, most of the coloured impurities phenol (311 v./v.).

having been removed bythe previous extraction procedures. 1

Before nitrous acid treatment the extract contained 665 gm. of vitamin B12 activity and 2.43 g. of solids, and after treatment it contained 650 gm. of vitamin B12 activity and 0.35 g. of solids. The vitamin B12 activities were determined by means of Lactobacillus lactis Dome? organism.

Example 2 25 ml. of a solution of active material obtained by partial purification of the broth obtained by growing the organism Streptomyces griseus was acidified to pH 4 with glacial acetic acid and 3 ml. of saturated sodium nitrite solution was added. The mixture was left at room temperature for minutes and was then extracted 3 times with equal volumes of butanol, the butanol being back-washed with a little water to ensure no loss of the active material. The aqueous solution was then taken to pH 7.3 by means of solid sodium bicarbonate, and extracted 3 times with butanol- The phenolic layer was diluted with 5 volumes of diethyl ether and the mixture extracted with small amounts of water. Finally, the combined water layer was extracted with diethyl ether to remove the last traces of phenol.

Before extraction the solution contained 487 agm. of vitamin B12 activity and 1.22 g. of solids; after the nitrous acid treatment and butanolphenol extraction procedure the solution contained 450 agm. of vitamin B12 activity and 0.76 of solids. The vitamin B12 activities were determined by means of the Lacto'bacillus Zactis Dorner organism.

Example 3 100 ml. of a liver side-fraction, containing 20.3 gm. of solids and 8000 gm. of vitamin B12 activity was treated with 4 gm. of sodium nitrite and concentrated hydrochloric acid to pH 4 and warmed gently for one hour. The solution was acidified further to pH 3 and extracted with 4 equal volumes of n. butanol. The extracts were back-washed with small volumes or" water, which were added to the main aqueous layer. This was then neutralised and extracted 3 times with 1 volumes of phenol-butanol mixture (1:2). The extracts were diluted with several volumes of ether and the activity recovered by extracting with small quantities of water.

The product contained only 1.77 gm. of solids and 4000 gm. of vitamin B12 activity.

Example 4 m1. of a solution of active material obtained by partial purification of the broth obtained by growing the organism Streptomyces griseus was acidified to pH l with glacial acetic acid and 3 mi of saturated sodium nitrite solution was added. The mixture was left at room temperature for 15 minutes and then the pH adjusted to 7.3 with solid sodium bicarbonate. This solution was then extracted 3 times with butanol-phenol (3:1 v./v.) and the phenolic layer diluted with 5 volumes of diethyl ether, the mixture being extracted with small amounts of water. Finally, the combined water layer was extracted with diethyl ether to remove the last traces of phenol.

Before extraction the solution contained 487 g. of vitamin B12 activity and 1.22 g. of solids; after nitrous acid treatment and butanol-phenol extraction procedure the solution contained 437 g. of vitamin B12 activity and 0.89 g. of solids.

6 The vitamin B12 activities were determined-by means of Lactobacillus lactis Domer organism.

Example 5 50 ml. of a solution of active material obtained by partial purification of the broth obtained by growing the organism Streptomyces griseus was acidfied to pH 4 with glacial acetic acid and 1.5 g. of sodium nitrite added in the form of a saturated solution. The mixture was heated to C. for 5 minutes and then left to cool for 20 minutes. The pH was adjusted to 7.3 with solid sodium bicarbonate and the solution extracted 3 times with butanol-phenol (3:1 v'./v.). The phenolic layer was diluted with 5 volumes of diethyl ether, and the mixture was extracted several times with small amounts of water. Finally, the combined aqueous layer was extracted with diethyl ether to remove the last traces of phenol.

The original solution before nitrous acid and butanol-phenol treatment contained 500 g. of vitamin B12 activity and 5.6 g. of solids; after the purification procedure the solution contained 450 g. of vitamin B12 activity and 1.47 g. of solids; The vitamin B12 activities were determined by means of the Lactobacillus Zac'tis Dorner' organism.

Example 6 300 cc. of crude liquors derived from the culture of S. griseus and containing 23.5 g. of solids and approximately 24 mg. of vitamin B12 activity, were heated to 70 C. at pH 4 in the presence of 12 g. of sodium nitrite, kept at 70 C. for 5 minutes and then cooled. The solution was adjusted to pH 7.5 and extracted with three equal volumes of a mixture of phenol and benzene (1:3). To the combined organic layers two volumes of benzene were added and the mixture extracted exhaustively with several portions of water. The combined aqueous extracts contained 5.15 g. total solids (i. e. about 22% of the original) and 20.1 mg. of vitamin B12 activity (i. e. about of the original).

Example 7 300 cc. of crude liquors derived from the culture of S. grz'seus and containing 23.5 g. of solids and approximately 24 mg. of vitamin B12 activity, were allowed to stand with 2.4 g. of sodium nitrite at room temperature at pH 4 for 20 minutes. The solution was then treated as in Example 6. The total solids of the final aqueous extracts were 3.4 g. '(i. e. 14.4% of the original solids); the total vitamin B12 activity in the extracts corresponded to 83% of the original total.

We claim:

1. A process for separating difficultly separable impurities from anti-pernicious anaemia active material, which comprises the steps of, treating an aqueous extract of said active material containing said impurities with nitrous acid so as to contact said impurities with said nitrous acid to thereby modfy said impurities and render them more easily separable from said active material, and thereafter separating anti-perm cious anaemia active material and modified im-, purities from each other.

2. The process defined in claim 1 in which, after treatment with nitrous acid, an alkali is added to said aqueous extract to render it nonacid and anti-pernicious anaemia active material is extracted from the resulting aqueous extract with an organic solvent for said. active material, which solvent is at'least partially immiscible with water.

3. The process defined in claim 1 in which the modified impurities are extracted from said aqueous extract after treatment with nitrous acid with an. organic solvent for such .impurities, which solvent is at least partially immiscible with water. v 4. In a process for separating difficultly separable impurities from anti-pernicious anaemia active material, the'steps' which comprise, treating an aqueous extract of said active material containing said impurities with nitrous acid so as to. contact said impurities with said nitrous acid to thereby modify said impurities and render them more easily separable from said active material, thereafter extracting modified impurities from the resulting aqueous extract with an organic solvent for such impurities, which solvent is at least partially immiscible with water, then adding an alkali to the remaining aqueous extract to render it non-acid, and extracting antipernicious anaemia active material from the nonacid aqueous extract with an organic solvent for said active material, which solvent is at least partially immiscible with water.

5. A process for the separationof difficultly separable impurities from anti-pernicious anaemia active material, which comprises the steps of, treating an aqueous extract of said active material containing said impurities with nitrous acid so as to contact said impurities with said nitrous acid to thereby modify said impurities and render them more easily separable from said active material, and extracting the modified impurities from the aqueous extract with ether, while said aqueous extract is at a pH of from 1 to 5.

6. The process defined in claim which includes the additional step of adding an alkali to the remaining aqueous extract to render it non-acid after the extraction of modified impurities therefrom, and thereafter extracting antipernicious anaemia active material from the nonacid aqueous extract with a solvent selected from the group consisting of phenol and mixtures of phenol and butanol.

7. A process for the separation of impurities containing amino groups from anti-pernicious anaemia active material, which process comprises the steps of, treating an aqueous extract of said active material containing said impurities with a nitrite at a pH of approximately 4 to destroy said amino groups and convert said impurities into hydroxy acids, and thereafter separating anti-pernicious anaemia active material and said hydroxy acids from each other.

8. The process defined in claim '7 in which said nitrite is selected from the group consisting of sodium nitrite and potassium nitrite.

9. A process for the separation of difiicultly separable impurities from anti-pernicious anaemia active material, which comprises the steps of, treating an aqueous extract of said active material containing said impurities with nitrous acid so as to contact said impurities with said nitrous acid to thereby modify said impurities and render them more easily separable from said active'material, and extracting the modified impurities from the aqueous extract with chloroform while said aqueous extract is at a pH of from 1 to 5.

10. The process defined in claim 9 which 111-.- cludes the additional step of adding an alkali to the remaining aqueous extract to render it nonacid after the extraction of modified impurities therefrom, and thereafter extracting anti-pernicious anaemia active material from the non-acid aqueous extract with a solvent selected from the group consisting of phenol and mixtures of phenol and butanol.

11. A process for the separation of difficultly separable impurities from anti-pernicious anaemia active material, which comprises the steps of, treating an aqueous extract of said active material containing said impurities with nitrous acid so as to contact said impurities with said nitrous acid to thereby modify said impurities and render them more easily separable from said active material, and extracting the modified impurities from the aqueous extract with butyl alcohol while said aqueous extract is at a pH of from 1 to 5.

12. The process defined in claim 11 which includes the additional step of adding an alkali to the remaining aqueous extract to render it nonacid after the extraction of modified impurities therefrom, and thereafter extracting anti-pernicious anaemia active material from the nonacid aqueous extract with a solvent selected from the group consisting of phenol and mixtures of phenol and butanol.

13. A process for the separation of difficultly separable impurities from anti-pernicious anaemia active material, which comprises the steps of, treating an aqueous extract of said active material containing said impurities with nitrous acid so as to contact said impurities with said nitrous acid to thereby modify said impurities and render them more easily separable from said active material, and extracting the modified impurities from the aqueous extract with amyl alcohol while said aqueous extract is at a pH of from 1 to 5.

14. The process defined in claim 13 which includes the additional step of adding an alkali to the remaining aqueous extract to render it nonacid after the extraction of modified impurities therefrom, and thereafter extracting anti-pernicious anaemia active material from the non-acid aqueous extract with a solvent selected from the group consisting of phenol and mixtures of phenol and butanol.

ERNEST LESTER SMITH. STANLEY BALL.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Name Date Keresztesy June 23, 1942 OTHER REFERENCES Number 

1. A PROCESS FOR SEPARATING DIFFICULTY SEPARABLE IMPURITIES FROM ANTI-PERNICIOUS ANAEMIA ACTIVE MATERIAL, WHICH COMPRISES THE STEPS OF, TREATING AN AQUEOUS EXTRACT OF SAID ACTIVE MATERIAL CONTAINING SAID IMPURITIES WITH NITROUS ACID SO AS TO CONTACT SAID IMPURITIES WITH SAID NITROUS ACID TO THEREBY MODFY SAID IMPURITIES AND RENDER THEM MORE EASILY SEPARABLE FROM SAID ACTIVE MATERIAL, AND THEREAFTER SEPARATING ANTI-PERNICIOUS ANAEMIA ACTIVE MATERIAL AND MODIFIED IMPURITIES FROM EACH OTHER. 